All viruses that contain class II or class III fusion proteins (and some with class I) fuse from within endosomes. For these viruses, the endosome is the initial site of infection. In response to the low-pH endosomal environment, a fusion protein undergoes conformational changes that cause merger of the viral and endosomal membranes, releasing the viral genetic material into cytosol. If regions on the fusion protein critical for infection are located, they will provide targets for new anti-viral drugs and vaccines. Properties of the endosomal membrane itself and its interior such as membrane voltage, acidic lipids, and redox potentials could also exert profound effects on fusion; if regulatory properties are identified and could be modified, new methods of halting infection could result. Voltage across endosomal membranes has been shown by our laboratory to control fusion of a number of types of virions that have class II or class III fusion proteins: the naturally occurring negative voltag across a membrane promotes fusion; positive voltage inhibits it. The universality of voltage dependence of class II and III fusion proteins is now reasonably certain. Chimera experiments strongly suggest the transmembrane domain (TMD) as the region of the fusion protein that confers voltage sensitivity. Voltage dependence could arise either because a TMD directly responds to voltage, or because acidic (negatively charged) lipids in outer membrane leaflets bind to TMDs. The concentration of acidic lipids in outer leaflets varies with voltage-dependent flip-flop between leaflets: enriching the concentration of acidic lipids in outer leaflets by experimentally incorporating them and measuring the consequences to voltage-dependent fusion will determine if acidic lipid binding causes voltage-dependent fusion. If it does, the binding region on the fusion protein will be identified by altering the protein. If the TMD is the voltage sensor, measuring displacement currents of synthetic TMDs will determine whether a large dipole moment is the key for sensing voltage. The energy required to transfer electrons (redox potentials) may also have an important role in regulating viral fusion: The redox potential of an endosome depends on its level of NADPH oxidase (NOX). Inhibition of NOX activity within endosomes indicates that the number of virions that fuse varies with the oxidation state in the same manner it does in cell-cell fusion. NOX activity will be altered, and fusion within endosomes monitored by confocal microscopy, to determine the relevance of redox potentials in infection. Methods to monitor membrane insertion of segments of viral fusion proteins will be developed through coupling lipophilic, charged probes to a fusion protein and electrophysiologically determining if voltage dependence of fusion is altered. This method will have far greater sensitivity than current methods. Class II and III proteins share some structural features; insertion studies could thus yield fundamental principles that unify the mechanisms of action of fusion proteins in these two classes. Clinically, identifying the mechanisms for endosomal control of viral fusion will reveal which processes could be interrupted to reduce or prevent infection.